JP2009137950A - Photoacoustic imaging agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a photoacoustic imaging agent comprising a particle as a light-absorbing ingredient, provided that the particle has a structure converting an input signal, which is a weak near-infrared light of within the MPE (maximum permissible exposure) that is scattered in vivo and reaches deep parts of a living body, into a large acoustic output signal. <P>SOLUTION: The photoacoustic imaging agent for use in photoacoustic tomography (PAT) diagnosis contains the particle as the light-absorbing ingredient, provided that the particle has a core part containing a crystal comprising an alkali halide or an alkaline earth halide, a shell part that covers the core part so that the core part does not come into contact with the external environment and a marker part that selectively reacts with a substance derived from a target disease to be detected. The average particle size of the entire particle comprising the core part, the shell part and the marker part is ≤100 nm. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a photoacoustic contrast agent used for photoacoustic tomography (PAT) diagnosis.

  Increasing diseases such as tumors and arteriosclerosis has become a major social issue.

  In order to diagnose the presence or absence of such diseases, measurement methods using invasive living body imaging devices such as X-ray CT and PET-CT have been put into practical use, but a less invasive measurement method is desired. It has been. Photoacoustic tomography is a candidate for a less invasive measurement method. Photoacoustic tomography is a noninvasive measurement method in itself (light irradiation itself). However, the difference in optical properties between a diseased part such as a tumor or arteriosclerosis and a normal part is not sufficiently large in detection of photoacoustic tomography. Therefore, in order to use photoacoustic tomography for the diagnosis of the presence or absence of a tumor or arteriosclerosis, it is necessary to administer a contrast agent to enhance the contrast. That is, diagnosis using photoacoustic tomography is a minimally invasive method. FIG. 2 shows a schematic diagram of a conventional biological imaging apparatus. The water tank 5 is filled with water and irradiates light 16 from a light source 7 onto a fixed object 6 to be measured. Light from the light source is irradiated through an optical system such as a mirror 8 and a concave lens 9. A pulsed acoustic signal emitted from the object to be measured when irradiated with light is collected by the transducer (Transducer) 10 through water, and the transducer is rotated about the center of the sample (object to be measured). A biological image is obtained by performing image processing on a signal obtained by scanning. Reference numeral 11 denotes an amplifier, 12 denotes an oscilloscope, 13 denotes a computer, 14 denotes a step motor, and 15 denotes an oscillation element. As a light source used for photoacoustics, near-infrared light having a high biological permeability in the range of 600 nm to 1300 nm is used.

  For conventional photoacoustic contrast, Non-Patent Document 1 can be referred to.

  In Non-Patent Document 1, indocyanine green (ICG) and indocyanine green stabilized with polyethylene glycol (ICG-PEG) are intravenously injected into rats as contrast contrast agents, and blood vessels in the head are imaged by photoacoustic imaging. The absorption spectrum of a contrast contrast agent is shown in Non-Patent Document 1. Further, the intensity of laser light that can be irradiated on the living body is within the maximum allowable exposure (MPE) as defined by JIS C 6802: 2005, and near-infrared light is scattered in the living body. The strength is weakened. Energy obtained by multiplying the intensity of light reaching the contrast agent by an absorption coefficient is absorbed by the contrast agent, partly causing molecular vibration of the contrast agent, and the vibration is converted into heat. The heat is retained inside the contrast medium, and part of it is propagated to the surrounding biological material, and these undergo microdeformation according to their thermal expansion coefficient, and sound is generated accordingly. As a result, a sound pressure as a photoacoustic signal is detected.

  The above-mentioned conventional photoacoustic contrast agent has the following problems.

First, since the light intensity in the deep part of the living body is weak, the heat generated by the contrast agent is as small as several millimeters K (in Non-Patent Document 1, the exposure intensity is ² mJ / cm ² and 2.6 mK). For this reason, the sound pressure as the photoacoustic signal detected in the deep part of the living body is inevitably small, and the contrast of the deep part of the living body of the obtained image is lowered.

  Second, near-infrared light has less energy than the wavelength in the ultraviolet region. Near-infrared light energy can cause molecular rotation and vibration, but it is less than the dissociation energy between the atoms that make up the molecule, so it cannot break the molecular bond, and more than the activation energy that causes a chemical reaction. Because of its small size, the reaction cannot be accelerated by light or the heat generated by turning the light.

Thirdly, as the concentration of the dye increases, concentration quenching, anisotropy of light absorption due to an increase in optical anisotropy in the aggregated state, polarization of light absorption, shift of light absorption wavelength, light Since phenomena such as saturation / decrease of absorption efficiency occur, the accumulation effect by increasing the density is limited to about 10 ¹⁷ per cm ³ .

On the other hand, substances having color centers are known. A substance having a color center is a colorless and transparent single crystal. However, it is colored when irradiated with primary excitation light such as γ-rays, X-rays, electron beams, ultraviolet rays, or when exposed to alkali metal vapor. Absorbs light in the wavelength range. Non-Patent Document 2 shows a light absorption spectrum of a substance having a color center.
“Nonvasive photoacoustic angiography of animal brains in vivo with near-infrared light and an optical contrast agent”, Xueding Wanget. , Optics Letters / Vol. 29, no. 7, pp. 730-732 / April 1,2004 “Spectral Location of the Absorption Due to Color Centers in Alkalidides Crystals”, HENRY f. IVEY, Physical Review, Vol. 72, no. 4, pp. 341-343, August 15, 1947

  In order to solve the above-described problems, the present invention has a structure capable of converting weak light that arrives while light below the MPE in the near-infrared wavelength region is scattered in the living body into an input signal and converts it into a large acoustic output signal. An object of the present invention is to provide a photoacoustic contrast agent having particles having a light-absorbing component.

The photoacoustic contrast agent according to the present invention for solving the above problems is
In a photoacoustic contrast agent used for photoacoustic tomography (PAT) diagnosis,
A core containing a crystal comprising an alkali halide or an alkaline earth halide;
A shell portion that covers the core portion so that the core portion does not touch the external environment;
A marker part that selectively reacts with a substance derived from a specific disease to be detected, and particles having a total average particle size of 100 nm or less including the core part, the shell part, and the marker part It contains as an absorption component, It is characterized by the above-mentioned.

  According to the photoacoustic contrast agent according to the present invention, it is possible to use weak light below the MPE in the near-infrared wavelength region as an input signal and convert it into a large acoustic output signal.

  Hereinafter, the photoacoustic contrast agent of the present invention will be described in detail. An example of the structure of the photoacoustic contrast agent of the present invention is schematically shown in FIGS. 1 (a) and 1 (b). The active ingredient of this photoacoustic contrast agent, that is, the particle as the light absorbing component is a crystal comprising an alkali halide or an alkaline earth halide (a crystal having both an alkali halide and an alkaline earth halide as the core portion 1). May be). The color center can be formed by irradiating the core part with primary excitation light or by exposing the core part to alkali metal vapor. A shell portion 2 is provided as an outer shell covering the core portion 1. Furthermore, a marker portion 3 is provided on the outer surface of the shell portion 2. A conjugate part 4 is provided between the shell part 2 and the marker part 3 to connect them as necessary. FIG. 1A shows an example in which the conjugate part 4 connects the shell part 2 and a separate marker part 3. FIG. 1B shows a modification in which the shell portion 2 is taken into the marker portion 3 or the shell portion 2 is included in the marker portion 3.

(Substance group capable of forming a color center and means for forming a color center)
Examples of the alkali halide or alkaline earth halide used in the photoacoustic contrast agent of the present invention include one or more of lithium, sodium, potassium, rubidium, cesium, francium, beryllium, magnesium, calcium, strontium, barium, and radium. A salt composed of a combination of a cation and one or more anions of fluorine, chlorine, bromine, iodine, and astatine can be used. Two or more salts may be mixed and used. It can also be used as a complex salt composed of a plurality of these cations and a plurality of anions. Further, a perhalogenate based on a combination of a perchlorate anion or a perbromate anion and an alkali or alkaline earth cation can also be used. In the present invention, a perhalogenate is also a kind of halide.

  Since the above substances generally have no optical absorption in a wide wavelength region from the visible wavelength region to the infrared region, the single crystal is usually colorless and transparent. On the other hand, when the above substances are irradiated with primary excitation light such as γ-rays, X-rays, electron beams, ultraviolet rays, or when exposed to alkali metal vapor, they become colored and absorb light in a specific wavelength range. Hereinafter, substances having such properties are collectively referred to as substances having a color center.

(Color-based basic physical properties)
The color center is described in the following known documents.
・ "Optical Property Handbook" (p.228, p.398)
・ "Color Centers in Alkali-Halides, and A11ied Phenomena" (Electronic Processes in Ionic Crystals, p. 109)
・ "Optical properties, electron-lattice interaction" (Solid physics separate volume, pages 17 and 82)
・ "Materials and light" (Science and Technology Basic Course 24, 130)
FIG. 3A and FIG. 3B are diagrams showing the coloring principle of a substance having a color center in terms of structure and energy.

FIG. 7 is a diagram illustrating a polymorph of the color center. In the figure, “circles marked with“ e ”or“ e ⁻ ”represent electrons,“ hatched circles ”represent cations,“ black circles ”represent holes, and“ white circles ”represent anions. It is known that the color center does not always take any one of the polymorphs shown in FIG. 7, but transforms (deforms). Since the color center only needs to be absorbed with respect to the light wavelength used in the photoacoustic contrast imaging of the present invention, the color center may be any shape among polymorphs. In addition to the localized state of electrons shown in FIG. 7, a hydrogen atom-like model in which electrons are actually delocalized has been proposed, but the color center of the present invention is in such a form. There may be. The fact that the color center can take such a hydrogen atom-like structure is said to be the reason why the color center has a large vibrator strength, that is, a large light absorption efficiency.

  It is understood that the coloring mechanism is due to the trapping of excitons composed of electron-hole pairs in the ionic crystal, as shown in FIGS.

  When the primary excitation light is irradiated, excitons are trapped in the crystal as a pair of “electrons” and “holes”. “Hall” cannot exist alone for a long time, but the “Halogen anion pair (−2 valence)” in which the atomic coordinates of two “halogen anions (eg, assumed to be −1 valence)” are biased by the impact of excitation A model (Vk type) in which “halogen anion pair + hole (−1 valence)” in which “hole (+1 valence)” is captured and stabilized is considered. The coloring when irradiated with the primary excitation light can be explained as follows. That is, in terms of energy, as shown in FIG. 3B, there is a (halogen) trap level between the conduction band and the valence band, and the primary excitation light changes from the valence band to the trap level. Electrons are excited. Hereinafter, this is referred to as primary excitation, and a state in which electrons are excited in the trap level is referred to as an excited state. This excited state has a characteristic that it has a relatively long lifetime at temperatures near room temperature.

  On the other hand, coloring when exposed to alkali metal vapor is said to be due to trapping of alkali metal as a pair of “alkali ion (alkali cation)” and “electron”. In this case, excitons consisting of pairs of “electrons” and “alkali cations” are trapped at the lattice points of the Schottky type, that is, the original crystal lacking “alkali cations” and “halogen anions”. It is thought. The electrons in this case are also in an excited state.

  In any of the above cases, the color center is in an excited state.

  By irradiating light in the absorption band to the color center in such an excited state, the trapped electrons can be photoexcited from the trap level to the conduction band (hereinafter referred to as secondary excitation). ). In the process in which electrons excited in the conduction band by secondary excitation make a transition to the ground state without radiation, large energy corresponding to primary excitation is released. That is, the color center can emit a large energy corresponding to the primary excitation light with a small energy of the secondary excitation light. Although FIG. 3A shows a NaCl type crystal lattice, excitons are similarly trapped in the case of a CsCl type crystal lattice, and a color center is formed.

(Correlation between color center absorption wavelength and core chemical composition)
The light absorption band of the color center formed in the crystal used for the core part can be adjusted by the combination of the ionic species of the substance group that can form the color center. That is, by selecting a combination of ion species of a substance group capable of forming a color center, a color center having absorption in the wavelength band of light used for photoacoustics can be formed in a crystal used for the core portion. Such selection can be performed using, for example, an absorption spectrum of a color center formed when a bromide salt and a chloride salt as shown in FIGS. 4 and 5 are irradiated with X-rays. Moreover, selection can be performed using the absorption spectra of bromide salts, chloride salts, fluoride salts, and iodide salts shown in Non-Patent Document 2 mentioned above. Examples of the material for forming the core include NaF, NaCl, NaBr, and NaI.

In addition, by using a mixed crystal in which these ionic species are mixed, the absorption spectrum can be continuously changed according to the mixing ratio, and a color center having a suitable absorption spectrum can be designed. Therefore, the present invention is characterized in that the mixed crystal composition has a color center absorption spectrum adapted to the wavelength band of light used for photoacoustics. For example, a salt in which 3 to 4 components of a plurality of cations (for example, A, B) and a plurality of anions (for example, C, D) are “mixed” (for example, A _0.5 B _0.5 C or A _{0. 5} B _0.5 C _0.5 D _0.5 , A _0.2 B _0.8 C _0.7 D _0.3, or the like) can be used.

  Furthermore, compared with the ICG broad near-infrared spectrum shown in the conventional example, the above-mentioned color center absorption spectrum peak (absorption spectrum peak at the time of secondary excitation) has a narrow wavelength band, and a laser is used. High effective quantum efficiency.

  In using the photoacoustic contrast agent of the present invention, it is preferable to use light in the near infrared region having a wavelength of 600 nm to 1300 nm for secondary excitation. This wavelength band called a “biological window” is considered to be used as a light irradiation light source in a living body because light absorption by blood hemoglobin is small and light absorption by water is also small.

  Since the present invention is a contrast agent, it is preferable to adjust the absorption wavelength at the color center so as to correspond to the wavelength of light used in the photoacoustic tomography apparatus, that is, light irradiated to the contrast agent.

  In the light irradiation to the dye shown in the conventional example, an expensive wavelength variable DYE laser is used. In contrast, since the contrast agent of the present invention can adjust the absorption wavelength, a general-purpose and inexpensive gas laser or laser diode can be used for light irradiation. The core part of the present invention can easily adapt the absorption peak at the color center to the laser wavelength. For example, if potassium bromide is used for the core, the absorption peak at the color center can be adapted to the laser wavelength of the helium-neon laser at 633 nm. As another example, by using a mixed crystal of potassium bromide and rubidium bromide in a molar ratio of 1: 9, an absorption peak having a color center at 680 nm, which is a laser wavelength of a laser diode developed for optical disks. Can be adapted.

(Shell part)
When the alkali halide (or alkaline earth halide) in the core of the present invention absorbs moisture and deliquesces, the crystal structure cannot be maintained. As is clear from the principle, the color center exists only in the crystalline material, and cannot exist in the solution material. Therefore, providing a shell portion that protects the core portion from water vapor in the atmosphere and moisture in the living body is an important feature when the color center is used as a contrast contrast agent. As the material used for the shell portion, a material that covers the core portion to form an outer shell, prevents the core portion from coming into contact with the external environment, and at least prevents moisture and water vapor from entering. Further, the term “coating” used herein means that the core portion is covered with the shell portion so as not to come into contact with moisture, water vapor, or the like existing in the external environment. Such a shell part can be formed, for example, by coating the outer surface of the core part or by modifying the outer surface of the core part. As the coating or modifying material, organic or inorganic polymer compounds are suitable. Examples of the organic polymer compound include various polymers, polypeptides and derivatives thereof, and two or more kinds of copolymers or organic dendrimer compounds thereof. Mixtures of two or more of these can also be used. Examples of the polymer include polyethylene glycol, polyvinyl alcohol, polylactic acid, poly (meth) acrylic acid and esters thereof, polyethylene, polystyrene, polyethylene terephthalate, gelatin, silicone, and polysiloxane. Examples of the inorganic polymer compound include silica and alumina. Moreover, the composite_body | complex which has either of the above as a main component can also be utilized. The shell is not limited to a polymer, and a low molecular weight lipid bilayer can be used as the shell. Further, for example, an organic-inorganic hybrid compound or a mixture of an organic compound and an inorganic compound can be used.

(Marker part and conjugate of marker part and shell part)
The marker portion has a configuration that selectively reacts with a substance derived from a specific disease to be detected. A marker for detecting various diseases such as tumor and arteriosclerosis can be preferably used for the marker portion. The marker generally has specific selective adsorption. Markers include antibodies that selectively react with antigens, enzymes that selectively react with substrates, and substances that contain a molecular structure that causes a specific adsorption reaction with the detection target in a low oxygen or low pH region. can do.

  In order to conjugate the marker part and the shell part, for example, a molecule having a carboxyl group at the end is used as the shell part described above, the carboxyl group is succinimide esterified, and the amino group and amide bond of the antibody or enzyme The technique of forming can be used. As another example, the end of the molecule constituting the shell can be maleimidized and condensed with a thiol group of an antibody or an enzyme for conjugation.

(Particle size)
As a result of primary excitation, the number of excitons captured in the crystal, that is, the number of color centers, depends on the wavelength, output, exposure time, defect density, impurity concentration, etc. of the primary excitation light. The upper limit is said to be 10 ¹⁸ per cm ³ . When the density of color centers is 10 ¹⁸ per cm ³ , the calculation is such that 1000 color centers exist in a cube with a side of 100 nm.

  Crystals having a fine particle diameter of several tens to 100 nm can be obtained from an alkali halide (or alkaline earth halide) aqueous solution by a spray drying method or an ink jet method. The spray drying method is a method in which an aqueous solution of alkali halide is sprayed to form fine droplets, and the droplets are exposed to dry air to crystallize as fine particles. The ink jet method is a method for obtaining fine particle crystals by discharging fine droplets from a fine nozzle and drying them or dropping them into a poor solvent with respect to an alkali halide such as butanol.

  It is preferable that the total particle size when the shell is formed on the outer side of the core and the final contrast agent is conjugated with the marker site is such that drug transfer (drug delivery) is possible in vivo. . In consideration of blood vessel permeability, the particle size ((the size of the particle as a whole including the core, the shell, and the marker portion: hereinafter referred to as the average particle size) is preferably 100 nm or less, More preferably, the average particle size is 100 nm or less, which can reduce the risk of thrombosis during administration to a living body. 100 nm or less is one standard because of the contribution of molecular structure and molecular higher order structure, etc. The particle size here means the particle size measured by the dynamic light scattering method.

(Color center formation timing)
The particles as an active ingredient for light absorption of the contrast agent according to the present invention can be stored and transported in a state in which the core part, the shell part, and the marker part are combined. The contrast agent of the present invention can be prepared from only particles as an active ingredient for light absorption or, if necessary, mixed with a carrier or a diluent. When using a contrast agent in which the color center has not been formed in advance, the core of the contrast agent is primarily excited and administered to the diagnosis target in the preparation stage of image acquisition by photoacoustic tomography (PAT). To do. At that time, as described above, the core portion is colored when irradiated with primary excitation light such as γ-rays, X-rays, electron beams, and ultraviolet rays, and a color center having light absorption in the wavelength band of light for PAT diagnosis is formed. . Since the shell part and the marker part almost transmit the primary excitation light, the excitation light is mainly absorbed by the core part.

  Hereinafter, the present invention will be described in more detail with reference to examples.

(Example 1) Production of photoacoustic contrast agent 0.5 g of potassium bromide (molecular weight 119.01) was dissolved in pure water so that the amount of the solution was 1 ml, and a 4.2 M aqueous solution was produced (at a saturated concentration). The concentration is close to 1 g / 1.5 ml). This potassium bromide aqueous solution was crushed and dispersed with pressurized air, and the generated mist was classified and mixed with dry air at room temperature to form fine-particle crystals having a particle size in the range of 60 nm to 100 nm. At that time, AP-9000G manufactured by Shibata Science Co., Ltd. was used as a particle generator. The average particle size was measured using a dynamic light scattering method.

  This potassium bromide fine particle crystal is dispersed in paraffin as a core part, and N-hydroxysuccinimide ester (NHS) -terminated polyethylene glycol (manufactured by NOF Corporation) with an average molecular weight of 12000 is added to adsorb around the core part. To form a core-shell structure. The core-shell structure thus obtained was extracted into an aqueous phase to obtain a water dispersion state. Thereafter, an antibody against a receptor expressed in mouse macrophages was labeled on the shell part, and the marker part was conjugated. When the average particle diameter of the particles was measured by a laser diffraction particle size distribution analyzer, the particle diameter was 70 nm to 100 nm. X-rays (Cu-Kα rays, 45 kV, 40 mA) were irradiated for 1 hour or more to the particles conjugated with this marker part, and it was confirmed that potassium bromide was colored blue and a color center was formed. This can be used as a photoacoustic contrast agent.

(Example 2) Diagnosis with photoacoustic contrast agent The whole amount of the photoacoustic contrast agent prepared in Example 1 is intravenously administered to a mouse in which a mouse macrophage receptor is expressed in the lung. 30 minutes later, a He—Ne laser pulse of 633 nm was irradiated at an intensity of 32 mJ / cm ² , and an acoustic signal from a receptor expression site in a mouse lung using a plurality of Immersed Transducers (trade name: manufactured by Toray Engineering) in water. Measure. At that time, at least the part of the mouse that contains the lungs is placed in water. The distance between the receptor expression site and the Immersion Transducer is estimated from the delay time from when the laser pulse is irradiated until the acoustic signal is observed as shown in FIG. 6, and the acoustic signal observed at several points around the mouse is coded. Tomography is obtained by image processing using a pattern method or the like. On the other hand, when no photoacoustic contrast agent was administered, no acoustic signal was observed even when irradiated with the same light as described above, and contrast was achieved by the specific accumulation of the photoacoustic contrast agent at the receptor expression site in the lung. I can confirm that.

(Example 3) Preparation of a photoacoustic contrast agent having an absorption peak at 680 nm 1 ml of a mixed aqueous solution containing 0.06 g of potassium bromide (molecular weight 119.01) and 0.74 g of rubidium bromide (molecular weight 165.39) Prepared. In this mixed aqueous solution, potassium bromide and rubidium bromide are present in a molar ratio of approximately 1: 9. Fine particle core formation, shell formation, and marker conjugate formation were performed by the same means as in Example 1 to prepare a photoacoustic contrast agent suitable for a near-infrared laser diode having a wavelength of 680 nm.

  According to the preferred embodiment of the present invention described above, a photoacoustic contrast agent having a structure capable of converting weak light below the MPE in the near-infrared wavelength region into an input signal and converting it into a large acoustic output signal. Obtainable.

(A) And (b) is a schematic diagram of an example of the photoacoustic contrast contrast agent of this invention. It is a schematic diagram of the conventional biological imaging apparatus. (A) And (b) is a figure explaining the color center formation mechanism used for the photoacoustic contrast agent of this invention. It is the figure which measured the absorption spectrum of the various ionic crystals which formed the color center. It is the figure which measured the absorption spectrum of the various ionic crystals which formed the color center. It is a photoacoustic signal when 532 nm light is irradiated to the color center of a sodium bromide crystal. It is a figure showing the polymorphism of a color center.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Core part 2 Shell part 3 Marker part 4 Conjugate part 5 Water tank 6 Measured object 7 Light source 8 Mirror 9 Concave lens 10 Transducer (Transducer)
11 Amplifier
12 oscilloscope 13 computer 14 step motor
15 Oscillator 16 Light

Claims (7)

In a photoacoustic contrast agent used for photoacoustic tomography (PAT) diagnosis,
A core containing a crystal comprising an alkali halide or an alkaline earth halide;
A shell portion that covers the core portion so that the core portion does not touch the external environment;
A marker part that selectively reacts with a substance derived from a specific disease to be detected, and particles having a total average particle size of 100 nm or less including the core part, the shell part, and the marker part A photoacoustic contrast agent comprising an absorbing component.   2. The photoacoustic contrast agent according to claim 1, wherein a wavelength band of light for the PAT diagnosis is a near infrared region of 600 nm to 1300 nm.   The photoacoustic contrast agent according to claim 1, wherein the shell portion contains an inorganic polymer compound.   The photoacoustic contrast agent according to claim 3, wherein the inorganic polymer compound is silica, alumina, or a composite containing these as a main component.   The photoacoustic contrast agent according to claim 1, wherein the shell portion contains an organic polymer compound.   The shell part contains, as the organic polymer compound, polyethylene glycol, a polypeptide and a derivative thereof, a copolymer of two or more of these, an organic dendrimer compound, or a mixture of two or more of these. The photoacoustic contrast agent according to claim 5.   The photoacoustic contrast agent according to any one of claims 1 to 6, wherein the marker part includes an antibody or a molecular structure that causes a specific adsorption reaction in a low oxygen region or a low pH region.

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